A Rapid and Reproducible Genomic DNA Extraction Protocol for Sequence-Based Identification of Archaea, Bacteria, Cyanobacteria, Diatoms, Fungi, and Green Algae
Saba، Farkhondeh نويسنده Microorganisms Bank, Iranian Biological Resource Center (IBRC), (ACECR), Tehran, Iran. Saba, Farkhondeh , Papizadeh، Moslem نويسنده Microorganisms Bank, Iranian Biological Resource Center (IBRC), (ACECR), Tehran, Iran. Papizadeh, Moslem , Khansha، Javad نويسنده Microorganisms Bank, Iranian Biological Resource Center (IBRC), (ACECR), Tehran, Iran. Khansha, Javad , Sedghi، Mahshid نويسنده Microorganisms Bank, Iranian Biological Resource Center (IBRC), (ACECR), Tehran, Iran. Sedghi, Mahshid , Rasooli، Mehrnoosh نويسنده Microorganisms Bank, Iranian Biological Resource Center (IBRC), (ACECR), Tehran, Iran. Rasooli, Mehrnoosh , Amoozegar، Mohammad Ali نويسنده , , Soudi، Mohammad Reza نويسنده Department of Biology, Faculty of Science, Alzahra University, Tehran, Iran , , Shahzadeh Fazeli، Seyed Abolhassan نويسنده Department of Molecular and Cellular Biology, Faculty of Basic Sciences and Advanced Technologies in Biology, University of Science and Culture, Tehran, Iran ,
Background: Sequence-based identification of various microorganisms including Archaea, Bacteria, Cyanobacteria, Diatoms, Fungi, and green algae necessitates an efficient and reproducible genome extraction procedure though which a pure template DNA is yielded and it can be used in polymerase chain reactions (PCR). Considering the fact that DNA extraction from these microorganisms is time consuming and laborious, we developed and standardized a safe, rapid and inexpensive miniprep protocol. Methods: According to our results, amplification of various genomic regions including SSU, LSU, ITS, β-tubulin, actin, RPB2, and EF-1 resulted in a reproducible and efficient DNA extraction from a wide range of microorganisms yielding adequate pure genomic material for reproducible PCR-amplifications. Results: This method relies on a temporary shock of increased concentrations of detergent which can be applied concomitant with multiple freeze-thaws to yield sufficient amount of DNA for PCR amplification of multiple or single fragments(s) of the genome. As an advantage, the recipe seems very flexible, thus, various optional steps can be included depending on the samples used. |